CD4 is a nonpolymophic transmembrane glycoprotein expressed on the surface of most thymocytes and a subpopulation of matured T cells (CD4+ T cells) (see Germain, Curr. Biol., 7: R640-R644 (1997)). It is composed of four immunoglobulin-like extracellular domains, a transmembrane segment and a cytoplasmic tail non-covalently associated with a src-family tyrosine kinase, Lck. As an important component of the immune system, CD4 functions as a co-receptor assisting the T cell receptor (TCR) on the CD4+ T cells for stronger association with class II major histocompatibility complex (MHCII) on antigen-presenting cells (APCs). The association is sufficient to trigger T-cell signaling transduction resulting in activation of the CD4+ T cells.
CD4 also contributes directly to the signal transduction by driving signaling cascade through its kinase-linked cytoplasmic tail. The crystal structure of human CD4-murine MHCII shows that only the first extracellular domain (D1) of CD4 makes contact with MHCII (see Wang et al., Proc. Natl. Acad. Sci. USA, 98: 10799-10804 (2001)). However, mutational analysis indicates that in addition to D1, other domains also affect binding to MHCII (see Moebius et al., Proc. Natl. Acad. Sci. USA, 90: 8259-8263 (1993)). Moreover, oligomerization of CD4 is required for stable interaction with MHCII and efficient T-cell activation (see Sakihama et al., Proc. Natl. Acad. Sci. USA, 92: 6444-6448 (1995)).
CD4 is also the primary receptor for HIV-1 (see Dalgleish et al., Nature, 312: 763-767 (1984)). HIV-1 entry is initiated by binding of the viral envelope glycoprotein (Env) gp120 to cellular receptor CD4. The interaction results in extensive conformational rearrangements of gp120 and subsequently gp41 after engagement of a coreceptor (either CCR5 or CXCR4). The structural rearrangements of Envs and the interplay between Envs and the cellular receptor and co-receptor bring viral membrane toward target cell membrane, and eventually cause membrane fusion and viral entry. Because CD4 plays a key role in HIV-1 infection, recombinant solubly expressed CD4 (sCD4) containing either all four (T4) (see Deen et al., Nature, 331: 82-84 (1988)) or the first two extracellular domains (D1D2) (see Traunecker et al., Nature, 331: 84-86 (1988)) is a potent inhibitor of HIV-1 entry and used for crystallization alone (see Wu et al., Nature, 387: 527-530 (1997) and Ryu et al., Nature, 348: 419-429 (1990)) or with gp120 (see Kwong et al., Nature, 393: 648-659 (1998)).
In Sharma et al. (Biochemistry, 44: 16192-16202 (2005)), D1 was generated by using a mutational strategy and purified from the sonication supernatant of E. coli. However, the purified protein was stable only at low pH (4.0) and partially improperly folded, and had affinity several-fold lower than that of D1D2 or T4.
In Chen et al. (J. Virol., 85: 9395-9405 (2011)), two stable monomeric D1 mutants, mD1.1 and mD1.2 were identified, which were significantly more soluble and bound Env gp120s more strongly (50-fold) than D1D2, neutralized a panel of HIV-1 primary isolates from different clades more potently than D1D2, induced conformation changes in gp120, and sensitized HIV-1 for neutralization by CD4-induced antibodies.
However, the desire for new D1s that are correctly folded, highly soluble and stable under physiological conditions while preserving not only binding activity and specificity but also other functions, such as induction of conformational changes in HIV-1 gp120, remains.